CDV和CPV及CCV多重纳米PCR检测方法的建立与应用  

Establishment and rudimentary application of the multiplex nano PCR(nano-mPCR) to the detection of CDV,CPV and CCV

作　　者：罗亚坤[1,2] 刘琪[1,2] 蔺文成[3] 王静[1,2] 周灵[1,4] 梁琳[1,4] 崔尚金[1,4] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]农业部兽用药物与兽医生物技术北京科学观测实验站,北京100193 [3]华南农业大学动物科技学院,广东广州510642 [4]吉林农业大学动物科学技术学院,吉林长春130118

出　　处：《中国兽医科学》2016年第10期1207-1212,共6页Chinese Veterinary Science

基　　金：中国农业科学院创新工程项目(ASTIP-IAS16)

摘　　要：参考Gen Bank上登录的犬瘟热病毒（CDV）N基因序列、犬细小病毒（CPV）VP2基因序列和犬冠状病毒（CCV）M基因序列的保守型片段分别设计特异性引物,建立了CPV（193 bp）、CDV（500 bp）和CCV（780bp）的多重纳米PCR（nano-mPCR）检测方法,同时考查所建立nano-mPCR检测方法的特异性和敏感性。结果表明,该方法特异性和敏感性良好,对CPV、CDV和CCV的最低核酸检测量分别为1.39×10~2、6.0×10~2和6.7×10~2copies/L,其敏感性比普通PCR高100倍。临床样品的检测结果表明,nano-mPCR的建立为CDV、CPV和CCV的单独或混合感染进行早期快速、灵敏、准确的鉴别诊断提供了新方法。According to the gene sequences in Gen Bank of CDV,CPV and CCV,three pairs of specific primers were designed for amplifying the three specific fragments of CPV VP2（193 bp）,CDV H（500 bp） and CCV M（780 bp）,respectively.After optimization of annealing temperature and primers concentrations,a multiplex nano PCR assay（nano-mPCR） was established for simultaneous detection of the three viruses.The sensitivity and specificity tests showed that the nano-mPCR was highly sensitive in that as little as 1.39×102copies/ L（CPV）,6.0×102copies/ L（CDV-H） and 6.7×102copies/ L（CCV-M）.No band was amplified from canine infectious hepatitis virus,canine parainfluenza virus,canine herpes virus,Escherichia coli by the nano-mPCR.Fifty-nine clinical samples were detected by the nano-mPCR.As a result,five samples（8.47%） were positive for CDV and CPV,two samples（3.38%） were positive for CCV.The nano-mPCR could be used to effectively detect and differentiate CPV,CDV and CCV single or co-infection in clinical samples.

关 键 词：多重纳米PCR 犬瘟热病毒 犬细小病毒 犬冠状病毒 检测 流行病学 

分 类 号：S852.655[农业科学—基础兽医学]